Polyhydroxyalkanoates (PHA) are polyesters synthesized by a variety of bacterial strains as intracellular carbon and energy storage compounds grown usually under stress conditions. As biodegradable and biocompatible materials, Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] was first found from Ralstonia eutropha cultivated with 4-hydroxybutyric or 4-chlorobutyric acid as carbon sources in 1988. The incorporation of 4HB units into P(3HB) improves the material application potentials, and the copolymers show a wide range of physical properties ranging from highly crystalline plastic to elastic rubber, depending on the polymer composition. P(3HB-co-4HB) with various 4HB compositions are promising materials that have favorable biodegradability and mechanical properties.
Generally, carbon sources structurally related to 4HB are required to generate 4HB-containing PHA, such as 4-hydroxybutyric acid, γ-butyrolactone and 1,4-. However, these carbon sources are much more expensive than glucose or other 3HB-generating carbon sources. For example, 4-hydroxybutyric acid is considered to be the most effective precursor for forming 4HB monomer, but it is difficult to obtain since 4-hydroxybutyric acid is a controlled substance in countries including USA and China. The high cost of raw material for the copolymer production has become an obstacle for the wide production and application of P(3HB-co-4HB).
There were attempts using engineered bacteria to produce P(3HB-co-4HB) with carbon sources that are structurally unrelated to 4-hydroxybutyrate. U.S. Pat. No. 6,117,658 utilizes the PHA biosynthetic pathway in combination with a succinic semialdehyde metabolic pathway to produce P(3HB-co-4HB) in a recombinant host. The host cell contains a recombinant nucleotide sequence encoding the PHA biosynthetic pathway, and another recombinant nucleotide sequence encoding the succinic semialdehyde metabolic pathway. However, 4HB monomer content in the P(3HB-co-4HB) is relatively low, from 1.5 to 5 mol %, while it is common regarded that P(3HB-co-4HB) may show desirable physical properties when the 4HB content in the polymer is higher than 10 mol %. The full content of U.S. Pat. No. 6,117,658 is incorporated herein as reference to the current specification.
There is a need in the art for constructs and methods of producing P(3HB-co-4HB) with high 4HB monomer content using carbon sources are structurally unrelated to 4-hydroxybutyrate in an efficient and cost-effective manner.